D values of Salmonella enteritidis isolates and quality attributes of shell eggs and liquid whole eggs treated with irradiation

D Values of Salmonella enteritidis Isolates and Quality Attributes of Shell Eggs and Liquid Whole Eggs Treated with Irradiation L. E. SERRANO,* E. A. MURANO,*,†,‡ K. SHENOY,† and D. G. OLSON*,‡ *Department of Animal Science, †Department of Microbiology, Immunology and Preventive Medicine, and ‡Department of Food Science and Human Nutrition, Iowa State University, Ames, Iowa 50011

(Key words: eggs, Salmonella enteritidis, irradiation, quality attributes) 1997 Poultry Science 76:202–205

process that can be used to solve this problem. Extensive research has shown that ionizing energy at medium doses can eliminate non-spore-forming pathogens such as Salmonella in food products (Radomyski et al., 1994). Irradiation also causes very little increase in food temperature during application; thus it is termed “cold processing”. These features make the process of irradiation more attractive for eliminating pathogens in heatsensitive products like eggs. Relatively few studies have been conducted to evaluate the effect of irradiation on the microbiological and sensory quality of eggs. Some of the studies on quality characteristics indicate that irradiation of eggs can adversely affect color and flavor (Morgan and Siu, 1957). However, Rauch (1971) showed that irradiation at levels sufficient to pasteurize eggs did not cause significant changes in appearance or functional properties of egg white proteins. The objectives of the present study were 1) to determine the effect of low dose irradiation on the survival of various Salmonella enteritidis isolates on the surface of whole shell eggs (WSE), 2) to determine the effect of irradiation on the survival of S. enteritidis isolates present in the yolk of WSE, 3) to determine the effect of heating followed by irradiation on S. enteritidis isolates inoculated into homogenized liquid whole eggs (LWE), and 4) to determine the effect of irradiation on the quality attributes of eggs as measured by color and protein denaturation analyses.

INTRODUCTION Salmonella enteritidis is a foodborne pathogen associated chiefly with eggs and egg-containing foods. Eggs have been incriminated as the source of 80% of the outbreaks in the U.S. between the years 1985 to 1989 (Centers for Disease Control, 1990). Eating of raw or undercooked eggs has been cited as the primary cause of human infection with this pathogen. Vertical and horizontal transmissions are the two ways by which eggs can become contaminated by Salmonella. In vertical transmission, infected ovaries and oviducts of the hen are the major sources of contamination. In such cases, the organisms are usually found inside the eggs (Humphrey et al., 1989; Hopper and Mawer 1988; Pearson et al., 1987). Eggs can become contaminated also on the surface, either from feces or the environment; this is usually referred to as “horizontal transmission”. Washing eggs with disinfectants helps in reducing Salmonella counts primarily from the shell surface. The only commercial process used presently to eliminate pathogens from inside the eggs is pasteurization. However, heat pasteurization can affect quality of the egg contents. Low dose irradiation is an alternate

Received for publication November 21, 1995. Accepted for publication September 20, 1996.

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inoculated (2.4 × 106 cells per milliliter) with two S. enteritidis isolates and were heat-treated at 50 C for 0, 20, 40, or 60 min followed by irradiation at 0, 0.25, 0.5, 0.75, or 1.0 kGy. The results indicate that mild heating prior to irradiation was ineffective in reducing the irradiation D values. However, on the basis of the D values obtained, an irradiation dose of 1.5 kGy should be sufficient to reduce Salmonella counts by approximately 4 log10 in both whole shell and liquid eggs. Results also indicate that color and thermal characteristics of the whole or liquid eggs were unaffected by a 1.5-kGy dose of irradiation.

ABSTRACT Irradiation sensitivity of five Salmonella enteritidis isolates inoculated either on the surface or inside of whole shell eggs were determined. The shell eggs were irradiated at doses of 0, 0.5, 1.0, and 1.5 kGy. A minimal dose of 0.5 kGy was sufficient to eliminate all the isolates from the surface of whole eggs; however, the same isolates were more resistant to irradiation when present inside the eggs. The ATCC 13076 isolate was significantly more sensitive to irradiation, with a D value of 0.32 kGy, than the other four isolates from animal origin. Irradiation D values of the latter ranged from 0.39 to 0.41 kGy. Liquid whole eggs were also

IRRADIATION OF SALMONELLA ENTERITIDIS ISOLATES IN EGGS

MATERIALS AND METHODS

Bacterial Cultures Five S. enteritidis isolates were used, with four being of animal origin (one bovine, one from snake, and two from chicken all obtained from the National Animal Disease Center1). The fifth isolate, ATCC 13076, was obtained from the American Type Culture Collection. The isolates were maintained on slants of tryptic soy agar2 at 5 C. Prior to inoculation into eggs, they were cultured separately in 460 mL of tryptic soy broth2 for 18 h at 37 C. The 18-h cultures were diluted in 0.1% peptone solution2 to achieve a concentration of 108 cells per milliliter in the initial inoculum.

Sample Preparation and Inoculation

1NADC, Ames, IA 50010. 2Difco Laboratories, Detroit, MI 48232-7058. 3Curwood Inc., Oshkosh, WI 54901. 4CVP Systems Inc., Downers Grove, IL 60515. 5 Becton Dickinson Microbiology System, Cockeysville,

MD 21030-0243. 6Model 400, Tekmar, Cincinnati, OH 45222. 7LI-7000 Datalogger, LI-COR, Inc., Lincoln, NE 68504-1395. 8MeV Industrie S.A., Jouy-en-Josas, Cedex, France. 9Briker Instruments, Inc., Billerica, MA 01821-3957.

barrier pouches, sealed under air, and irradiated as described before. Inoculation of Liquid Egg. Only two isolates (ATCC 13076 and snake) were used in this part of the study. Twenty WSE cleaned and sanitized with 70% ethanol were individually broken under aseptic conditions and the egg contents were placed separately in sterile stomacher bags. The contents were homogenized in a Stomacher Lab Blender6 for 2 min. The homogenates were transferred to sterile individual glass test tubes (25 × 76 mm size; 40 mL per tube) and placed in a 50 C water bath. The temperature at the center of the tubes was monitored by an iron-constantan thermocouple connected to a datalogger.7 On reaching the target temperature of 50 C, 1.0 mL of the bacterial culture (ATCC 13076 or snake isolate) was inoculated to achieve a final concentration of approximately 2.4 × 106 cells per milliliter in each tube. The contents in the tubes were vortex-mixed for uniform distribution of the inocula and placed back in the water bath as quickly as possible. Tubes were removed randomly from the water bath at regular intervals of 0, 20, 40, and 60 min and the contents were transferred to the high barrier pouches. The bags were sealed and irradiated as described before. One Salmonella isolate per experiment was studied when inoculating LWE to avoid overcrowding of tubes in the water bath, as this affected temperature of tube contents.

Irradiation Samples were irradiated at the Iowa State University Linear Accelerator Facility that houses a MeV CIRCE III Linear Electron Accelerator.8 Whole shell eggs were irradiated at room temperature by x-rays and LWE were irradiated by electron beam because of the differences in density and thickness of the samples. Whole shell eggs inoculated either on the surface or in the inside were irradiated at 0, 0.5, 1.0, or 1.5 kGy (two eggs per isolate per dose). The required doses were achieved by adjusting the power level (1 to 3 kW) and the conveyor speed (feet per second, short or long exposure time) of the irradiator. Dosimeters consisting of alanine pellets were placed on the top and bottom of the WSE. The pellets were then examined with an Electron Paramagnetic Resonance (EPR) spectrophotometer9 to measure the actual absorbed irradiation dose in the eggs. For the experiments involving inoculated LWE, the samples were first heated at 50 C for 0, 20, 40, or 60 min prior to irradiation. The sealed pouches containing the samples were then irradiated at 0, 0.25, 0.5, 0.75, and 1.0 kGy. Alanine pellets placed on the surface and bottom of the pouches were used to measure the actual absorbed dose as described before.

Microbial Analyses Following irradiation, the WSE or LWE were transferred to Stomacher bags to which sterile 0.1% peptone solutions were added (1:10 dilution) and mixed

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Medium grade A eggs were purchased for the entire study from a local grocery store in Ames, Iowa. The eggs were first washed by submersion in water at room temperature, and then sanitized using 70% ethanol for 2 min. Sanitized eggs were air dried and inoculated by three different methods: Surface inoculation of WSE, internal inoculation of WSE, and inoculation of homogenized LWE. Surface Inoculation. Forty sanitized WSE were surface inoculated by dipping them separately in any one of the five Salmonella inocula (108 cells per milliliter; 8 eggs per isolate) for 20 min. They were then transferred to a sterile rack for air drying at room temperature. This resulted in approximately 106 cfu/mL of Salmonella on the surface of each egg. The inoculated eggs were then individually packaged in high barrier pouches (O2 permeability of 3 to 4 cc per 100 in2 per 24 h at 22.8 C and 0% RH; Curlon (Grade 861)3 and sealed under air (Model A300 CVP machine).4 The packaged samples were placed in a single layer in an egg holding cardboard flat and irradiated. Inside Inoculation. Forty clean WSE were aseptically inoculated by injecting 1 mL of 108 cells per milliliter of Salmonella culture (8 eggs per isolate) using a sterile syringe with 22-gauge needle.5 The cultures were inoculated into the yolk through the broad end of the egg using a candler. The shell at the inoculation site was covered with sterile tape to prevent leakage of egg contents or contamination of the eggs by other organisms. The inoculated eggs were then packaged individually in high

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SERRANO ET AL. TABLE 2. Irradiation D values of Salmonella enteritidis isolates in liquid eggs following heat treatment at 50 C at various time intervals1

TABLE 1. Effect of x-ray irradiation on the survival of Salmonella enteritidis isolates inside whole shell eggs1 Isolate

D value

ATCC 13076 Bovine Chicken-1 Chicken-2 Snake

(kGy) 0.32 ± 0.40 ± 0.39 ± 0.41 ± 0.41 ±

0.015a 0.019b 0.018b 0.02b 0.02b

a,bD values with no common superscript differ significantly (P < 0.05). 1Experiment

was performed in triplicate (n = 3).

thoroughly in a Stomacher Lab Blender7 for 2 min. After making the appropriate serial dilutions in 0.1% peptone, the samples were surface plated in duplicate on tryptic soy agar plates. Salmonella enteritidis colonies were enumerated after incubation at 37 C for 24 h.

Differential Scanning Calorimetry. Uninoculated WSE and LWE without homogenization were packaged in high barrier pouches as described in the experiment conducted with inoculated eggs. They were then irradiated at dose levels of 0, 0.25, 0.5, 0.75, 1.0, and 1.5 kGy (three WSE and three LWE per irradiation dose). Following irradiation, the albumen was separated from the yolk in both WSE and LWE. The albumen was passed through a Buchner funnel seven times to reduce viscosity (Ma et al., 1990). Thermal protein denaturation characteristics of the less viscous albumen were then determined using a Differential Scanning Calorimeter (DSC)10 as described by Ma et al. (1990). Briefly, 10 m-L aliquots of the albumen were pressure sealed in DuPont aluminum hermetic pans and examined by DSC. The calorimeter was connected to a Perkin Elmer UNIX base data acquisition and control system10 to obtain thermograms. Samples were heated from 30 to 100 C with 10 C increase in temperature per minute. The denaturation temperature (Td) and the heat of transition was then calculated from the thermograms. Color Analyses. Three uninoculated WSE and LWE were also packaged and irradiated as described above. In both cases, the separated egg whites and yolks were then examined by HunterLab Labscan Spectrophotometer11 to determine the Hunter L, a, and b values.

Irradiation D values ATCC 13076

0 20 40 60

0.27 0.24 0.23 0.21

± ± ± ±

10Perkin Elmer DSC7 differential scanning calorimeter and database system, Norwalk, CT 06850. 11Hunter Associated Laboratories, Inc., Reston, VA 22090.

0.013 0.016* 0.011* 0.010*

0.38 0.37 0.35 0.33

± ± ± ±

0.018 0.018 0.017 0.016*

1Heating at 50 C for various time intervals followed by irradiation was performed in triplicate (n = 3). 2Heat treatment at 50 C for 0, 20, 40, and 60 min. *Significantly (P < 0.05) from the D value at time 0 in the same column.

eggs per replicate per inoculation). One Salmonella isolate per experiment was examined in LWE studies. The experiments were performed in triplicate using a total of 120 eggs for the two isolates. DSC and Color Analyses. Fifty-four WSE and another 54 LWE were used in the study. Three eggs were irradiated per dose followed by analysis with DSC10 to determine thermal protein denaturation of the albumen or were examined under the HunterLab Labscan spectrophotometer11 to determine L, a, and b values. The experiments were performed three times for both DSC and color analyses and the average values calculated. The microbiological, DSC, and color analysis data were analyzed by using SAS (SAS Institute, 1986). Significant differences in irradiation D values for the Salmonella isolates following inoculation into either WSE or LWE were determined by analysis of variance. Tukey’s multiple comparison procedure was applied to determine significant differences among the average DSC and color analyses values.

RESULTS AND DISCUSSION

Irradiation Sensitivity of S. enteritidis Isolates in WSE and LWE In the present study, S. enteritidis isolates were inoculated in three different ways in eggs, and their TABLE 3. Differential scanning calorimeter values of egg whites after irradiation of whole shell eggs at various doses

Statistical Analyses Inoculation Studies. Forty WSE were used per experiment for inoculation of the five Salmonella isolates either on the surface or inside the eggs. Each experiment was replicated three times using a total of 240 eggs (40

Snake isolate (kGy)

Temperature of denaturation1 Dose

Peak 1 (Conalbumin)

(kGy) 0 0.25 0.50 0.75 1.00 1.50

65.5 65.7 65.7 65.7 65.9 66.3

1Mean

Peak 2 (Ovalbumin) (C)

± ± ± ± ± ±

3.3 3.3 3.3 3.3 3.3 3.2

of three replicates (n = 3).

79.1 79.1 79.2 79.1 79.3 79.4

± ± ± ± ± ±

4.0 4.0 3.9 3.9 4.1 4.1

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Determination of Thermal Protein Denaturation and Color Characteristics of Eggs Following Irradiation

Heat treatment at 50 C

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IRRADIATION OF SALMONELLA ENTERITIDIS ISOLATES IN EGGS TABLE 4. Differential scanning calorimeter values of egg whites after irradiation of liquid whole eggs at various doses

min, respectively. Heating for the extended periods of time (40 and 60 min) may have resulted in the isolates becoming more sensitive to irradiation, resulting in slightly lower D values. However, as mentioned earlier, the decrease in D value was very small and the required heating time was at least 40 min, making the process of heating prior to irradiation not very feasible and of little commercial value. On the basis of the D values obtained in this study (Tables 1 and 2), we can conclude that a low dose of 1.5 kGy is sufficient to reduce Salmonella counts in both WSE and LWE by approx. 4.0 log10. The S. enteritidis counts in unprocessed, naturally contaminated eggs are very low and do not normally exceed 10 to 100 cfu/mL (Humphrey et al., 1991; Gast and Beard, 1992; Hopper and Mawer, 1988). Thus, a dose of 1.5 kGy should be sufficient to eliminate this pathogen from eggs.

Temperature of denaturation1 Dose

Peak 1 (Conalbumin)

(kGy) 0 0.25 0.50 0.75 1.00 1.50

64.9 65.0 64.9 64.8 64.8 64.4

1Mean

Peak 2 (Ovalbumin) (C)

± ± ± ± ± ±

3.2 3.3 3.2 3.2 3.2 3.2

78.2 78.2 78.2 78.3 78.4 78.4

± ± ± ± ± ±

3.9 3.9 3.8 3.9 3.9 4.0

of three replicates (n = 3).

Effect of Irradiation on Protein Denaturation and Color Characteristics of Eggs Differential scanning calorimetry is a rapid, sensitive, and convenient method to detect changes in the thermolability of the egg white protein. This procedure has therefore been used in the egg industry for evaluating egg quality. The thermograms of albumen from fresh eggs at the natural pH of 8.4 usually show two major endotherms (peaks): one at 65 and the second at 79 C produced by the denaturation of conalbumin and ovalbumin, respectively. Any changes or shifts in these endotherms indicate that proteins present in egg white are denatured and quality attributes altered, resulting in a lower quality product. In our study, the endotherms of egg white proteins of irradiated eggs as determined by DSC did not differ from those of nonirradiated eggs, thus showing that irradiation at the doses tested did not alter the thermal characteristics of egg white proteins (Tables 3 and 4). Significant differences (P > 0.05) in color for both egg whites and yolks were not observed between irradiated and nonirradiated samples in both WSE and LWE, regardless of the dose tested (Tables 5 and 6). Ma et al. (1990) have reported fading or discoloration of yolk color following irradiation at 2.98 kGy. However, our results

TABLE 5. Color characteristics of whole shell eggs following irradiation at various doses1 Egg white Dose

‘L’ value

(kGy) 0 0.25 0.50 0.75 1.00 1.50

14.2 15.9 14.4 17.3 16.5 19.1

1Mean

± ± ± ± ± ±

5.9 2.3 3.3 4.1 3.0 6.1

‘a’ value 0.7 0.7 0.5 0.7 0.7 0.5

of three values (n = 3).

± ± ± ± ± ±

0.2 0.3 0.3 0.4 0.3 0.4

Egg yolk ‘b’ value 1.4 1.3 1.3 1.8 0.7 1.1

± ± ± ± ± ±

0.8 0.7 0.6 0.8 0.5 0.7

‘L’ value 58.3 59.7 57.7 57.4 59.5 58.1

± ± ± ± ± ±

2.3 3.9 2.8 3.0 4.0 3.8

‘a’ value 2.2 3.4 1.3 2.3 1.7 2.6

± ± ± ± ± ±

1.1 1.2 1.0 1.0 1.2 1.4

‘b’ value 25.3 26.0 22.7 24.8 25.4 25.3

± ± ± ± ± ±

1.5 2.1 2.3 1.9 1.8 2.0

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sensitivities to low dose irradiation were determined. The effect of mild heat treatment (50 C) prior to irradiation of LWE was also examined. When inoculated on the surface of the eggs, a minimal dose of 0.5 kGy was sufficient to reduce the microbial counts of all the Salmonella isolates to undetectable levels of less than 102 cfu/mL (data not shown). However, the same Salmonella isolates were more resistant to irradiation when present inside whole eggs (Table 1). The ATCC 13076 isolate was found to be significantly (P > 0.05) more sensitive to irradiation, with a lower D value than those of the other isolates (Table 1). The difference in sensitivity of all the Salmonella isolates to irradiation when present on the surface vs when inside the eggs may be due to a protective mechanism involving the egg proteins and lipids. Heating has been shown to make Salmonella typhimurium more sensitive to irradiation in eggs (Licciardello, 1964). Mild heating of LWE followed by irradiation was therefore performed to determine if a combination of these two treatments would result in reduction and elimination of S. enteritidis using lower doses of both processing conditions. Irradiation D values in LWE for the ATCC and snake isolate following heat treatment at 50 C for various time intervals are given in Table 2. Although a significant difference (P > 0.05) does exist after heating for 40 or 60 min for the two isolates vs irradiation without heat treatment (at 0 min, see Table 2), it is not large enough to be of any practical importance. A 0.8 and 0.6 log10 decrease in survivors was observed when the ATCC and snake isolates were heated at 50 C in LWE for 40 and 60

206

SERRANO ET AL. TABLE 6. Color characteristics of liquid whole eggs following irradiation at various doses1 Egg white Dose

‘L’ value

(kGy) 0 0.25 0.50 0.75 1.00 1.50

18.5 20.0 18.9 18.1 20.3 20.4

1Mean

± ± ± ± ± ±

3.1 3.0 2.6 2.8 2.5 2.6

‘a’ value 0.6 0.5 1.1 0.6 0.6 0.5

± ± ± ± ± ±

0.4 0.3 0.4 0.3 0.3 0.3

Egg yolk ‘b’ value 0.9 1.2 2.2 0.9 1.4 1.2

± ± ± ± ± ±

0.6 0.4 0.7 0.6 0.4 0.5

‘L’ value 56.5 57.0 53.5 56.9 55.0 54.4

± ± ± ± ± ±

3.1 2.9 2.8 3.0 2.5 2.7

‘a’ value 1.8 1.8 1.3 1.9 1.3 2.0

± ± ± ± ± ±

0.7 0.7 0.6 0.7 0.5 0.7

‘b’ value 24.6 24.3 23.1 24.4 23.6 23.5

± ± ± ± ± ±

0.8 0.9 0.9 0.9 0.8 0.7

of three values (n = 3).

REFERENCES Centers for Disease Control, 1990. Update: Salmonella enteritidis infections and shell eggs — United States, 1990. Morbid. Mortal. Weekly Rep. 39:909–912. Gast, R. K., and C. W. Beard, 1992. Detection and enumeration of Salmonella enteritidis in fresh and stored eggs laid by experimentally infected hens. J. Food Protect. 55:152–156.

Hopper, S. A, and S. Mawer, 1988. Salmonella enteritidis in a commercial layer flock. Vet. Rec. 123:351. Humphrey, T. J., A. Baskerville, H. Chart, and B. Rowe, 1989. Infection of egg laying hens with Salmonella enteritidis PT4 by oral inoculation. Vet. Rec. 125:531–532. Humphrey, T. J., A. Whitehead, A.H.L. Gawler, A. Henley, and B. Rowe, 1991. Numbers of Salmonella enteritidis in the contents of naturally contaminated hen’s egg. Epidemiol. Infect. 106:489–496. Licciardello, J. J., 1964. Effect of temperature on radiosensitivity of Salmonella typhimurium. J. Food Sci. 29:469–474. Ma, C. Y., M. R. Sahasrabudhe, L. M. Poste, V. R. Harwalkar, J. R. Chambers, and K.P.J. O’Hara, 1990. Gamma irradiation of shell eggs. Internal and sensory quality, physicochemical characteristics, and functional properties. Can. Inst. Food Sci. Technol. J. 23:226–232. Morgan, B. H., and R.G.H. Siu, 1957. Action of ionizing radiation in individual foods. Pages 268–294 in: Radiation Preservation of Food. S. D. Bailey, J. M. Davies, B. H. Morgan, R. Pomerantz, R.G.H. Siu, and R. G. Tischer, ed. U.S. Government Printing Office, Washington, DC. Pearson, J. G., G. Southam, and R. A. Holley, 1987. Survival and transport of bacteria in egg washwater. Appl. Environ. Microbiol. 6:248–261. Radomyski, T., E. A. Murano, D. G. Olson, and P. S. Murano, 1994. Elimination of pathogens of significance in food by low-dose irradiation: A review. J. Food Prot. 57:73–86. Rauch, W., 1971. Influence of ionizing radiation on egg quality. Archiv. Geflu¨gelkd. 35:112–115. SAS Institute, 1986. SAS User’s Guide: Statistics. SAS Institute Inc., Cary, NC.

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indicate that a dose of 1.5 kGy did not cause any adverse changes in egg white or yolk. It is possible that a threshold dose exists, above which significant changes can be detected. Further studies to determine this dose may not be necessary if irradiation of eggs is limited to a maximum of 1.5 kGy. In conclusion, based on the D values reported in this study, a minimal dose of 0.5 kGy would be sufficient to eliminate S. enteritidis from the surface of whole eggs, and a dose of 1.5 kGy would be sufficient to eliminate the organism from WSE and LWE. Mild heating at the temperatures tested prior to irradiation was unnecessary, as it did not decrease radiation resistance of S. enteritidis significantly. Finally, thermal characteristics of egg white and the color of yolk and whites were not adversely affected by an irradiation dose of 1.5 kGy. Thus, the process of irradiation offers an advantage over heating alone, in that it can eliminate S. enteritidis from eggs without adversely affecting egg quality.